Nitrogen fixation by immobilized Azotobacter chroococcum

A nitrogen-fixing bacterium, Azotobacter chroococcum, was immobilized in 2% agar gel. The optimum partial oxygen pressure, pO₂, of immobilized cells was 0.2 atm, wherea s that of native cells was 0.05 atm. When continual nitrogen fixation was performed under aerobic conditions, the nitrogenase activity of immobilized cells increased with increasing time. On the other hand, the activity of native cells decreased rapidly. Increase of nitrogenase activity was attributed to growth of the bacteria in the gel matrix. The production rate of total nitrogen compounds by the immobilized bacteria was also increased during the first 4 days. Nitrogen compounds produced by the immobilized cells were mainly amino acids such as γ-aminobutyrate, glutamate and arginine.     

Cloning and characterization of hydrogenase genes from Azotobacter chroococcum

Summary Genomic DNA from Azotobacter chroococcum was shown by DNA hybridization to contain sequences homologous to Rhizobium japonicum H₂-uptake (hup) hydrogenase genes carried on the plasmid pHU1. Two recombinant cosmid clones, pACD101 and pACD102, were isolated from a gene library of A. chroococcum by colony hybridization and physically mapped. Each contained approximately 42 kb of insert DNA with approximately 27 kb of overlapping DNA. Further hybridization studies using three fragments from pHU1 (6 kb HindIII, 6.4 kb BglII and 5 kb EcoRI) showed that the hup-specific regions of R. japonicum and A. chroococcum are probably highly conserved. Weak homology to the hydrogenase structural genes from Desulfovibrio vulgaris (Hildenborough) was also observed. A 24 kb BamHI fragment from pACD102 subcloned into a broad host-range vector restored hydrogenase activity to several Hup^- mutants of A. chroococcum.     

Production of polyhydroxyalkanoates by Azotobacter chroococcum H23 in wastewater from olive oil mills (alpechin)

We describe the production of large amounts of homo- and copolymers of polyhydroxyalkanoates (PHAs) by Azotobacter chroococcum strain H23 when growing in culture media amended with alpechin. A. chroococcum grown on NH₄⁺-medium supplemented with alpechin formed PHAs up to 50% of the cell dry weight after 24h. The results show that alpechin supports the growth of strain H23 and also that this waste could be utilized as a carbon source. Production of PHAs by using alpechin looks promising, since the use of inexpensive feed-stocks for PHAs is essential if bioplastics are to become competitive products.     

Cloning of the gene for phosphoenolpyruvate carboxylase from Azotobacter chroococcum, an enzyme important in aerobic nitrogen fixation

Summary Azotobacter chroococcum Fos 189 is a Tn1-induced mutant which, unlike the parent strain MCD1, does not fix nitrogen in air when provided with glucose or pyruvate as sole carbon sources. Fos 189 showed 5% of parental activity for phosphoenolpyruvate carboxylase though PEP synthetase activity was normal. The A. chroococcum phosphoenolpyruvate carboxylase (ppc) gene was isolated after complementation of an appropriate Escherichia coli mutant using a broad host range gene bank prepared from A. chroococcum genomic DNA. The gene was localised by transposon mutagenesis and subcloning on a minimum DNA fragment of 6.6 kb. Broad host range plasmids containing the A. chroococcum ppc gene complemented the mutation in Fos 189 thereby restoring aerotolerant nitrogen fixation.     

Establishment of phosphate-solubilizing strains of Azotobacter chroococcum in the rhizosphere and their effect on wheat cultivars under green house conditions

A pot experiment was conducted in the green house to investigate the establishment of phosphate solubilizing strains of Azotobacter chroococcum, including soil isolates and their mutants, in the rhizosphere and their effect on growth parameters and root biomass of three genetically divergent wheat cultivars (Triticum aestivum L.). Five fertilizer treatments were performed: Control, 90 kg N ha^—1, 90 kg N + 60 kg P₂O₅ ha^—1, 120 kg N ha^—1 and 120 kg N + 60 kg P₂O₅ ha^—1. Phosphate solubilizing and phytohormone producing parent soil isolates and mutant strains of A. chroococcum were isolated and selected by an enrichment method. In vitro phosphate solubilization and growth hormone production by mutant strains was increased compared with soil isolates. Seed inoculation of wheat varieties with P solubilizing and phytohormone producing A. chroococcum showed better response compared with controls. Mutant strains of A. chroococcum showed higher increase in grain (12.6%) and straw (11.4%) yield over control and their survival (12—14%) in the rhizosphere as compared to their parent soil isolate (P4). Mutant strain M37 performed better in all three varieties in terms of increase in grain yield (14.0%) and root biomass (11.4%) over control.     

Chemical modification by trinitrobenzenesulfonate of a lipid and proteins of intracytoplasmic membranes isolated from Chromatium vinosum and Azotobacter vinelandii

1. 1. The structure of intracytoplasmic membranes of a photosynthetic bacterium Chromatium vinosum and a nitrogen-fixing bacterium Azotobacter vinelandii was studied by chemical modification of amino groups of phospatidylethanolamine and proteins with trinitrobenzensulfonate.

2. 2. Almost all the constituents of intracytoplasmic membranes of C. vinosum were solubilized in a mixture of chloroform, methanol and trichloroacetic acid. One-third of proteins in the intracytoplasmic membranes of C. vinosum was found solubilized in a mixture of chloroform and methanol. By using a column chromatography with Sephadex LH-20 in organic solvents, the unmodified as well as the trinitrophenylated proteins and also the trinitrophenylated phosphatidylethanolamine were separated from the other colored substances.

3. 3. In the chemical modification of the intracytoplasmic membrane preparations, 30% of phosphatidylethanolamine and 15% of protein amino groups in C. vinosum and 45% of phosphatidylethanolamine and 20% of protein amino groups in A. vinelandii were estimated to be exposed to the aqueous phase. In the single-layered liposomes composed of phosphatidylethanolamine and phospatidylglycerol with a ratio of 2:1, 40% of phosphatidylethanolamine were estimated to be exposed to the aqueous phase.

EPR studies by Fe isotopic substitution on the nature of an unknown electron acceptor in Azotobacter vinelandii phosphorylating particles

1. EPR ⁵⁷Fe isotopic substitution studies provide unequivocal evidence that the g = 2.011 signal found in oxidized Azotobacter vinelandii phosphorylating particles is due to an iron-containing structure. The broadening constant determined as a result of this electron—nuclear hyperfine interaction was 15.7 G.

2. A similar signal found in a number of iron—sulfur containing proteins was found by quantitative EPR estimations to exist in a variable but substantial concentration when compared to the intensity of the reduced g = 1.9 type EPR resonance.

3. Reaction of the phosphorylating particles with excess potassium ferricyanide resulted in an alteration of the initial g = 2.011 iron signal resulting in the detection by microwave power studies of at least two different iron species which exhibited major g-values at 1.992 and 2.027.     

The nifH, nifM and nifN genes of Azotobacter vinelandii: Characterisation by Tn5 mutagenesis and isolation from pLAFR1 gene banks

Summary Tn5 was introduced into Azotobacter vinelandii on a suicide vector, pGS9. Three Nif^- mutants were found to carry Tn5 in nifH (MV6), in nifN (MV22), and in or near nifM (MV21), from the results of hybridisation experiments. For MV21 and MV22 this was also shown by complementation with the nif genes of Klebsiella pneumoniae on pRD1. MV6 failed to synthesis the nifH, D and K gene products. MV6 and MV22 fixed nitrogen in the absence of supplied molybdenum while mutant MV21 did not, suggesting that the nifM gene product may be required for the alternative nitrogenase system synthesised in azotobacteria under conditions of molybdenum deprivation. Reconstitution experiments with mutant extracts showed that MV22 (nifN ^-) lacked the FeMo cofactor and that MV21 (NifM^-) synthesised inactive Fe protein. These biochemical phenotypes are identical to those of the K. pneumoniae nifN and nifM mutants, respectively, demonstrating that these genes have the same function in both K. pneumoniae and A. vinelandii. Complementation of the A. vinelandii mutants with pLAFR1 gene banks of A. vinelandii or a. chroococcum yielded three cosmids of interest. pLV10 complemented UW91, a nifH mutant, and corrected the defect in MV6 after recombination with the mutant genome. It also carried nifD (but not nifK) and about 18 kb of DNA upstream from nifH. pLV1 from the A. vinelandii gene bank complemented both MV21 and MV22 as did pLC11, isolated from the A. chroococcum gene bank. Both pLV1 and pLC11 carried part of the nif cluster downstream of nifHDK which also includes nifEN and nifMVS on about 22 kb of DNA.     

Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants

Azospirillum isolates were obtained from rhizosphere soil and roots of three cactaceae species growing under arid conditions. All Azospirillum isolates from rhizosphere and roots ofStenocereus pruinosus andStenocereus stellatus were identified asA. brasilense; isolates of surface-sterilized roots fromOpuntia ficus-indica were bothA. brasilense andA. lipoferum. Azospirilla per g of fresh root in the three species ranged from 70×10³ to 11×10³. The most active strains in terms of C₂H₂ reduction (25–49.6 nmol/h·ml) and indoleacetic acid (IAA) production (36.5–77 μg/ml) were those identified asA. brasilense and isolated from Stenocereus roots.A. lipoferum isolated from Opuntia roots produced low amounts of IAA (6.5–17.5 μg/ml) and low C₂H₂-reduction activity (17.8–21.2 nmol/h·ml).     

Pyruvate and ethanol as electron donors for nitrite reduction by Escherichia coli K12

Pyruvate and ethanol were both effective electron donors for nitrite reduction by Escherichia coli K12. The pyruvate-dependent rate decreased by approximately 50% when either a cysG mutation, which results in loss of NADH-dependent nitrite reductase activity (EC 1.6.6.4), or a chl mutation, which results in loss of the formate-nitrite oxidoreductase activity, was introduced into the prototrophic parental strain CGSC4315. A double mutant deficient in both of these previously described activities retained only 2% of the rate of nitrite reduction of the parental strain after growth on glucose or 5% after growth on pyruvate. We conclude that any third pathway for nitrite reduction contributes little to the in vivo rate of nitrite reduction by wild-type strains.     

Uranium reduction and microbial community development in response to stimulation with different electron donors

Stimulating microbial reduction of soluble U(VI) to less soluble U(IV) shows promise as an in situ bioremediation strategy for uranium contaminated groundwater, but the optimal electron donors for promoting this process have yet to be identified. The purpose of this study was to better understand how the addition of various electron donors to uranium-contaminated subsurface sediments affected U(VI) reduction and the composition of the microbial community. The simple electron donors, acetate or lactate, or the more complex donors, hydrogen-release compound (HRC) or vegetable oil, were added to the sediments incubated in flow-through columns. The composition of the microbial communities was evaluated with quantitative PCR probing specific 16S rRNA genes and functional genes, phospholipid fatty acid analysis, and clone libraries. All the electron donors promoted U(VI) removal, even though the composition of the microbial communities was different with each donor. In general, the overall biomass, rather than the specific bacterial species, was the factor most related to U(VI) removal. Vegetable oil and HRC were more effective in stimulating U(VI) removal than acetate. These results suggest that the addition of more complex organic electron donors could be an excellent option for in situ bioremediation of uranium-contaminated groundwater.     

欧美107杨树提取物体外对植物病原真菌的抑制活性

制备了欧美107杨树枝条和叶的乙醇粗提物及不同极性溶剂的萃取部分,并测定了它们对植物病原真菌的抑制活性。枝条和叶的乙醇粗提物对棉花枯萎病菌、小麦纹枯病菌、番茄早疫病菌、番茄枯萎病菌、黄瓜枯萎病菌、小麦赤霉以及玉米弯孢等7种植物病原真菌均具有一定的抑制作用。而枝条的乙醇粗提物对杨树溃疡病菌的菌丝生长有一定的促进作用。抗菌活性成分主要集中在乙酸乙酯萃取部分。     

Release of phosphodiesterase activator from particulate fractions of cerebellum and striatum by putative neurotransmitters

The endogenous phosphodiesterase activator (PDEA) described by Cheung (1,2) is, in part, stored as a membrane-bound protein (12,13). PDEA can be released from the membranes by a cAMP-dependent phosphorylation of a protein that may function as PDEA binding site (13). We found that PDEA can be released from brain particulate fraction by 1 M norepinephrine, dopamine, adenosine, and histamine in the presence of ATP and a purified cAMP-dependent protein kinase; in similar conditions, serotonin is ineffective in concentrations up to 0.1 mM. Norepinephrine and dopamine activate the adenylate cyclase activity of those preparations from which they release the PDEA. Norepinephrine is more potent than dopamine in releasing PDEA from the particulate fraction of cerebellum, whereas dopamine is more active than norepinephrine in releasing PDEA from the particulate fraction of striatum. The release of PDEA elicited by both neurotransmitters is concentration-dependent; increasing the transmitter concentrations above a certain limit decreases the rate of PDEA release.     

Host specificity of Rhizobium strains isolated from nitrogen-fixing trees and nitrogenase activities of strain GRH2 in symbiosis with Prosopis chilensis

Host plant specificity was examined in symbiosis between Rhizobium strains isolated from legume-tree root nodules and herbaceous or woody legumes from which they were isolated. Strain GRH2 isolated from Acacia cyanophylla formed effective nodules on Acacia, Prosopis and Medicago sativa as well. Nitrogenase activity, measured as acetylene reduction, of strain GRH2 in symbiosis with Prosopis chilensis was the highest (P 0.05) among the tropical legumes studied and was similar to those found for other associations involving herbaceous legumes. Relative efficiency of nitrogenase varied from 0.3 to near 1 during the light time of the photoperiod. However no hydrogen uptake activity was detected by the amperometric method used. Rhizobium strains GRH3, GRH5 and GRH9 isolated from A. melanoxylon, P. chilensis and Sophora microphylla, respectively, also showed a very low host-range specificity. All isolates were infective and effective on at least one of the herbaceous legumes tested. These data demonstrate the lack of specificity of Rhizobium strains isolated from nitrogen-fixing tree root nodules and that these strains can form effective nodules on herbaceous legumes.     

Spindle formation and cleavage in Xenopus eggs injected with centriole-containing fractions from sperm

The eggs of Xenopus laevis are capable of initiating spindle formation and cleavage in response to microinjection of partially purified components of sea urchin sperm. High activity was assayed from a sperm head fraction obtained after removal of the plasma and nuclear membranes, acrosome, midpiece mitochondrion, and tail. Chromatin, the basal plate, and the distal centriole comprised the components of the head fraction. Disruption of the chromatin did not impair activity and purified chromatin lacked activity, suggesting the centriole and basal plate as the active materials. Low doses of active material induced apparently normal cleavage at 90 min after injection, with 16% of the eggs reaching the late blastula stage. High doses of active material induced precocious multiple cleavage, with some eggs cleaving into 3–10 segments within 20 min after injection. These eggs contained numerous spindles and asters in the animal hemisphere, as judged by light microscopy of stained sections. Microinjection of eggs is presented as a semi-quantitative bioassay for agents initiating spindle formation and cleavage.     

Morphological and physiological changes in Leishmania promastigotes induced by yangambin, a lignan obtained from Ocotea duckei

We have previously demonstrated that yangambin, a lignan obtained from Ocotea duckei Vattimo (Lauraceae), shows antileishmanial activity against promastigote forms of Leishmania chagasi and Leishmania amazonensis. The aim of this study was to determine the in vitro effects of yangambin against these parasites using electron and confocal microscopy. L. chagasi and L. amazonensis promastigotes were incubated respectively with 50 μg/mL and 65 μg/mL of pure yangambin and stained with acridine orange. Treated-parasites showed significant alterations in fluorescence emission pattern and cell morphology when compared with control cells, including the appearance of abnormal round-shaped cells, loss of cell motility, nuclear pyknosis, cytoplasm acidification and increased number of acidic vesicular organelles (AVOs), suggesting important physiological changes. Ultrastructural analysis of treated-promatigotes showed characteristics of cell death by apoptosis as well as by autophagy. The presence of parasites exhibiting multiples nuclei suggests that yangambin may also affect the microtubule dynamic in both Leishmania species. Taken together our results show that yangambin is a promissing agent against Leishmania.